Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood brain barrier (BBB) function. Using human brain tissues and primary human brain endothelial cells (BMVEC) we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 or a novel resorcinol-based compound O-1966, greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical post-capillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in-vitro with barrier forming primary BMVEC. Remarkably, the addition of CB2R agonist increased trans-endothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of ICAM-1 and VCAM-1 surface expression in BMVEC exposed to various pro-inflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.
Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. Here we provide evidence that METH alters BBB function via direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human microvascular endothelial cells (BMVEC) to METH diminishes tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing expression of cell membrane associated tight junction (TJ) proteins. These changes were accompanied by enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Anti-oxidant treatment attenuated or completely reversed all tested aspects of METH induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.
Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.
Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.
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